Purification, characterization, and cloning of enamel matrix serine proteinase 1.
The maturation of dental enamel succeeds the degradation of organic matrix. Inhibition studies have shown that this degradation is accomplished by a serine-type proteinase. To isolate and characterize cDNA clones encoding this proteinase, we used two degenerate primer approaches to amplify part of the coding region using polymerase chain-reaction (PCR). First, we purified the proteinase from porcine transition-stage enamel matrix and characterized it by partial protein sequencing. The enzyme was isolated from the neutral soluble enamel extract by successive ammonium sulfate precipitations, hydroxyapatite HPLC, reverse-phase HPLC, DEAE ion exchange, and affinity chromatography with a Benzamidine Sepharose 6B column. The intact protein and lysylendopeptidase-generated cleavage products were characterized by amino acid sequence analyses. Degenerate oligonucleotide primers encoding two of the polypeptide sequences were synthesized. In a complementary strategy, degenerate oligonucleotide primers were designed against highly conserved active-site regions of chymotrypsin-like proteinases. Both approaches yielded PCR amplification products that served as probes for screening a porcine enamel organ epithelia-specific cDNA library. The longest full-length clone is 1133 nucleotides and encodes a preproprotein of 254 amino acids. We designate this protein enamel matrix serine proteinase 1 or EMSP1. The active protein has 224 amino acids, an isotope-averaged molecular mass of 24.1 kDa, and an isoelectric point of 6.0. Multiple-tissue Northern analysis indicates that EMSP1 is a tooth-specific protein. Gelatin enzymography shows a dramatic increase in EMSP1 activity in the transition-stage enamel matrix. EMSP1 is most homologous to kallikriens and trypsins.
Simmer
J P
JP
University of Texas Health Science Center at San Antonio, School of Dentistry, Department of Pediatric Dentistry 78284-7888, USA.
Fukae
M
M
Tanabe
T
T
Yamakoshi
Y
Y
Uchida
T
T
Xue
J
J
Margolis
H C
HC
Shimizu
M
M
DeHart
B C
BC
Hu
C C
CC
Bartlett
J D
JD
D000566:Amelogenesis; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003743:Dental Enamel / Q000201:enzymology*; D003746:Dental Enamel Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D007610:Kallikreins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013552:Swine
D003746:Dental Enamel Proteins; D007610:Kallikreins; D012697:Serine Endopeptidases; C120801:kallikrein 4